Well there is my idea of how to present my dissertation. I’m not sure if/where I should put my discussion on open notebook science. Also there are a couple things that I could see going elsewhere. I could describe the yeast and e. coli stuff in parallel instead of one after another. Also the HD exchange stuff could easily go right after the yeast, e. coli, or even the tobacco seed stuff. What to do…

Otherwise I think the story is pretty compelling: history of D2O and the unanswered question by Lewis. Investigations into D2O effects and trying to understand low D2O concentration effects, effects on macromolecules, and the understanding of large volume/long-term HD exchange.

Any feedback you may have would be GREATLY appreciated. I’ll send you a figshare t-shirt, or if you are XL, I’ll send you a hoodie (but I only have one).

Like this:

I got some awesome new data to show. The first is the compilation of all the Repeating Crumley experiments. And the second is some new data that I’ve been meaning to create and now have with the help of Koch.

Tobacco seed germination rates

The data above is the compilation of all the RC data. Each trial had different water types, but I combined the samples that were the same in every set (DDW, DI water, 33% D2O, 66% D2O, and 99% D2O). Steve adapted his R-code that applies binomial confidence intervals to a data set and used it on this data. If that makes no sense, then just know that the dotted lines are the most probable range of germination rates. For instance, in 66% D2O there is a ~70% likelihood that seeds will germinate at a rate within the dotted yellow lines.

Now it’s time for some brand new data:

tobacco seed root length

Here we went through the pictures from Trial 5 and compared the growth rates of the roots. We calculated the lengths of various seeds in each image and tracked the changes from image to image. We chose DI, DDW, and 1% D2O, because the D2O concentrations are relatively similar and because we wanted to test a hypothesis from a while ago. It’s interesting that the seeds in DDW and D2O grow at the same rate, while seeds in DI water grow at roughly half the speed.

H-D exchange (or D-exchange as I’ve sometimes referred to it) has been a problem I’ve dealt with in the lab for some time. It is essentially something that I need to minimize but can never actually stop. It is also a process that I know almost nothing about other than it happens, it occurs somewhat instantaneously, but may be mediated by evaporation rates (when dealing with DDW and 99% D2O). Now I’m perusing the internet looking for some information. Come take a walk with me:

At ScienceOnline 2013, I met a person who pointed me in the direction of a Dr. Richard Zare. She told me he was very knowledgable in the field and so I looked up his papers. He had fivepapersrelevantto exchange reactions, all of which are way above my head. So I’ll start on Wikipedia and work my way up.

For those unaware, hydrogen-deuterium exchange is a reaction where a covalently bonded hydrogen is replaced with a deuterium atom. In the case of my experiments this happen with water. If I have deuterium depleted water and I keep it in contact with the environment (which has deuterium in it at around 16mM), eventually it will reach equilibrium and the deuterium level (of the DDW) will rise. The mechanism that causes this, I presume, is D-exchange.

Apparently the reaction is pH dependent. It can be quenched around pH 2.6, but seems to work best at pH 7.0-8.0. That’s really interesting to me. The pH levels that I’m normally working around are optimal for these reactions. Unfortunately there is nothing I can do about that.

The reaction may also be quite slow. According to Wikipedia, exchange is slow/unlikely intramolecularly, but is quite rapid via exposed surface hydrogen bonds. For the purposes of NMR, in vivo deuterium incorporation would be valuable and is hard to attain by dissolving proteins in D2O.

The first person to measure H-D exchange was Kaj Ulrik Linderstrøm-Lang, and I would read some of his stuff right now but they are locked down. The mechanism he used to study D-exchange was pretty interesting and involved a density gradient tube. If I read this correctly, KULL filled a long tube with oils of different desities. He could place a drop of water, with a small amount of proteins in the drop, into the oils to determine the density. As reactions occur, the density would change and the drop would move accordingly.

Ok I’ve reached the limit of what I can do tonight without using the UNM network for access to papers. I’ll try to look for more information this weekend. The quest for a basic understanding of H-D exchange continues…